In vivo imaging of human malignant mesothelioma grown orthotopically in the peritoneal cavity of nude mice

Abstract

Malignant mesothelioma (MM) causes significant morbidity and mortality in patients. With increasing efforts devoted to developing therapeutics targeting mesothelioma, a xenograft mouse model with in vivo tumor imaging is especially desired for evaluating anti-tumor therapies. In the present study, we fluorescently labeled the NCI-H226 human mesothelioma cell line by a lentiviral vector harboring a luciferase-GFP (Luc/GFP) fusion gene driven by the RNA polymerase II promoter. After single-cell cloning by flow cytometry, a clone (named LMB-H226-GL) that stably expresses high levels of Luc/GFP was obtained. The in vivo tumorigenicity of Luc/GFP-labeled LMB-H226-GL was determined by using intraperitoneal injections of the cells in nude mice. LMB-H226-GL was found to be able to consistently form solid tumors in the peritoneum of mice. Tumor growth and aggressive progression could be quantitated via in vivo bioluminescence imaging. The model exhibited the pathological hallmarks consistent with the clinical progression of MM in terms of tumor growth and spread inside the peritoneal cavity. To evaluate the in vivo efficacy of drugs targeting mesothelioma, we treated mice with SS1P, a recombinant immunotoxin currently evaluated in Phase II clinical trials for treatment of mesothelioma. All the tumor-bearing mice had a significant response to SS1P treatment. Our results showed that this is a well-suited model for mesothelioma, and may be useful for evaluating other novel agents for mesothelioma treatment in vivo.

Keywords: H226/NCI-H226; immunotoxin; in vivo bioluminescence imaging; malignant mesothelioma; mesothelin; monoclonal antibody; mouse xenograft model; preclinical model.

Fig 1

In vitro characterization of LMB-H226-GL cells. A. FACS analysis showing GFP fluorescence in the LMB-H226-GL cell line. Shaded area: parental NCI-H226; heavy solid line: LMB-H226-GL. B. Mesothelin expression on LMB-H226-GL. Shaded area: NCI-H226 without anti-mesothelin staining; thin dotted line: LMB-H226-GL without staining; heavy solid line: NCI-H226 with staining; heavy dotted line: LMB-H226-GL with staining. C. WST-8 assay of NCI-H226 and LMB-H226-GL. Various amounts of SS1P immunotoxin were used to treat NCI-H226 and LMB-H226-GL cells.

Fig 2

In vivo imaging of LMB-H226-GL tumors in nude mice. A. High-dosage group, each mouse received 10 million cells. Animals were imaged the following day (d2) and then once every week thereafter. B. Low-dosage group, each mouse received five million cells and were imaged following the same schedule as the high-dosage group.

Fig 2

In vivo imaging of LMB-H226-GL tumors in nude mice. A. High-dosage group, each mouse received 10 million cells. Animals were imaged the following day (d2) and then once every week thereafter. B. Low-dosage group, each mouse received five million cells and were imaged following the same schedule as the high-dosage group.

Fig 3

In vivo tumor growth curve. A. Tumor growth was measured by bioluminescence photometry. The day when the mice were injected with the tumor cells was set as d1. The photometry of the in vivo imaging was accumulated by software Living Image 3.1.0 (Caliper Life Sciences) and the resulting photon counts were plotted as the Y-axis. Error bars represent the standard deviation from measurement of four mice in each corresponding group. At the end of the study, mice were sacrificed; tumors were immediately removed and measured by weight, size and fluorescence. Association of photon intensity with tumor weight (B) or size (C) was also performed. A stronger correlation between bioluminescence and tumor weight is shown (correlation coefficients are 0.77 for weight and 0.57 for size).

Fig 4

Necropsy and histological observance. A. Mesothelioma in peritoneal cavity. Arrow indicates the tumor mass. B. H&E staining (20x). C. Mesothelin IHC (20x) with membrane staining. D. GFP IHC (20x) with cytoplasmic staining.

Fig 5

SS1P treatment. Each mouse was i.p. injected with 10 million cells. At day 1 (treatd1) or day 9 (treatd9), the mice were given the first SS1P treatment by i.p. injection at a dose of 0.4 mg/kg body weight. The treatments were performed every other day as indicated by arrows. *Indicates the significant (p < 0.05) difference between treatment and control by using the one-way ANOVA statistical test (GraphPad Prism 5.03).